Recent advances at Caltech have introduced the possibility of Ultrafast Electron Microscopy (UEM), wherein an ultrafast (pico-femtosecond) laser pulse is used to liberate bursts of electrons from the source filament which are then used to image specimens with exposure times 9 or more orders of magnitude faster than normal. Because the principal resolution limitation in modern single particle analysis today is beam-induced specimen movement, we hypothesize that if we record images faster than large macromolecules can move appreciably, the resulting images will be dramatically sharper. Improved image sharpness will lead directly to more precise alignments, more powerful classification of heterogeneous populations, and higher resolution reconstructions. Towards that end, we will characterize beam-induced specimen movement as a function of exposure time through ~13 orders of magnitude (10+1 to 10-12 s). Because electrons repel each other, however, ultrafast electron bursts spread while traveling down the microscope column, degrading coherence. Thus we will also characterize by theoretical calculation and direct experimental measurement how short the bursts can be and still maintain adequate coherence. These two pieces of information should allow us to find that exposure time which optimally balances reduced specimen movement against decreased coherence to deliver the sharpest possible images. Using this optimal exposure time, we will record thousands of "single particle" images of an ideal "control" complex, the 20S proteasome, to demonstrate the merits of biological UEM. The culmination of this project will then be to determine an unknown structure of a complex of major importance. If successful, this advance could make structure determination to near-atomic resolution routine for large macromolecular complexes.